31 results about "Selectable marker" patented technology

Levels of expression of antibiotic resistance genes are increased up to six-fold by inserting a proteasome-targeting tag into transgenes expressed in eukaryotic cells. Various selectable marker proteins are combined with different destabilization domains, leading to up to 70% increase in transgene expression. The increase in expression varies highly depending on the engineered construct and the lines cells used. Increase in expression drives exosome loading of cargo proteins in some aspects. By increasing expression and by editing trafficking signals of cargo proteins, proteins that normally locate to the ER can be trafficked to exosomes. This disclosure discloses efficient exosome delivery of a wide variety of engineered proteins, including modified antigen proteins of SARS-CoV-2 and influenza, and other proteins such as a modified alpha galactosidase A, an extracellular domain of vascular endothelial growth factor fused to a constant region of a human immunoglobulin heavy chain, and modified trastuzumab heavy and light chains.

Dual vector systems and these comprehensive mammalian host cells are provided for the expression of multi-chain molecules, with each vector employing a different promoter controlling the expression of a selectable marker. The mammalian host cells can be used to generate a recombinant protein of interest in a mammalian cell culture.

Dual vector systems and mammalian host cells comprising these dual vector systems are provided, wherein each vector employs a different promoter that drives the expression of a selectable marker. A mammalian host cell can be used to produce a recombinant protein of interest in mammalian cell culture.

Herein is reported a nucleic acid comprising in operably linked form a nucleic acid encoding a selection marker, a nucleic acid encoding a self-cleaving peptide sequence, and a nucleic acid encoding a proteinaceous protease inhibitor. Further reported are methods for the recombinant production of a heterologous polypeptide using said nucleic acid as well as a cell comprising said nucleic acid according to the invention. Likewise reported is the use of the nucleic acid according to the invention for increasing the amount of the recombinantly produced heterologous polypeptide by reducing protease cleavage.

Provided are methods for inserting a large gene / polynucleotide between about 4.5Kb and about 8Kb and lacking a selection marker into a target genomic position in a cell by delivering the gene / polynucleotide by two AAV vectors in the presence of a p53-binding protein 1 (53BP1) inhibitor and a DNA-dependent protein kinase catalytic subunit (DNA- PKcs) inhibitor. The disclosed methods improve large gene insertion by at least 10-fold and obviate the need for a selection marker in the vector design, thereby providing more room in the AAV vector for gene delivery.

The application discloses a dCAPS molecular marker for detecting a residual amount of a cucumber fruit myclobutanil and application thereof, and relates to the technical field of molecular markers.The dCAPS molecular marker is derived from a SNP at a position of 158 of a CsABCI19 gene promoter.The dCAPS molecular marker is applied to detection of a residual amount of a cucumber fruit myclobutanil.The method comprises the following steps: (1) extracting DNA; (2) performing a PCR reaction; and (3) performing Nco I enzyme cutting, so that two band types are obtained, one is 491 bp, corresponding to SNP A, and the other is 444 bp and 48 bp, corresponding to SNP G, and the two types are identified as a fruit low myclobutanil residual amount germplasm and a fruit high myclobutanil residual amount germplasm respectively.The verification result of the dCAPS molecular marker in the germplasm resource shows that the identification result of the molecular marker is consistent with the phenotype, and the dCAPS molecular marker can be used for rapidly identifying the cucumber germplasm resource with a low myclobutanil residual amount in a fruit, and is a good breeding auxiliary selection marker.

The invention discloses an SNP molecular marker related to cottonseed oil content and application of the SNP molecular marker, and belongs to the technical field of molecular marker development. The SNP molecular marker is located at the 43284845th site of an upland cotton genome D03 chromosome, the nucleotide is A or G, the upstream and downstream sequences are shown as SEQ ID NO.1, and the G allele is associated with the high oil content character. The SNP molecular marker can be directly applied to (1) identification and screening of high-oil cotton germplasm resources: high-oil materials are rapidly screened from existing varieties or strains; (2) cotton molecular breeding practice: as an effective selection marker, the cotton molecular marker is used for guiding the breeding of filial generations, and by selecting individuals carrying G alleles (especially GG type), the directionality and success rate of breeding new varieties / lines of high-oil cotton are remarkably improved. The method has very important practical application value.

The disclosure provides, for example, a method of making a composition comprising purified DNA molecules. In some embodiments, the method comprises contacting a solution comprising a plurality of circular, double-stranded DNA molecules to a hydrophobic interaction chromatography matrix. In some embodiments, the circular, double-stranded DNA molecules lack one or both of a bacterial origin of replication and a selectable marker.

The invention provides a DNA marker plasmid as well as a combination system and application thereof, and relates to the field of molecular biology. The DNA marker plasmid is formed by connecting a linearized skeleton sequence and an insertion fragment sequence; the linearized skeleton sequence is derived from a reference plasmid from which a lactose operon sequence is deleted, and a selectable marker gene of the linearized skeleton sequence has a preset recognition site of restriction enzyme; the insertion fragment sequence is formed by connecting a plurality of DNA fragments in series, and recognition sites of restriction endonuclease are arranged between the adjacent DNA fragments. According to the plasmid, a restriction enzyme cutting site is introduced into a selective marker, so that a skeleton is directly used as a characteristic band, and the complete utilization of a sequence is realized. Waste and purification are avoided after enzyme digestion, the cost is remarkably reduced, and the preparation efficiency is improved.

The present invention provides host cells and vector systems that allow precise tuning of expression of selectable marker expression and increased integrated copy number of product genes so that host cell growth and expression of desired proteins or nucleic acid products from the host cell can be optimized.

A marker for gene recombination of Starmerella bombicola and a method for using the same. The present invention provides an adenine auxotrophic selection marker consisting of a gene encoding a phospho

A lentivlral vector for real-time monitoring of cell state which includes a regulatory element that regulates expression of a selection marker, wherein the regulatory element comprises cell-state-specific histone H3K27-acetylation marks and / or chromatin accessibility. Methods of identifying a cell- state-specific regulatory element in a cell; optically screening a compound for the ability to modify cell identity; in vivo labeling normal and / or diseased cells at a specific developmental stage or a specific stage of disease; and real- time monitoring of transcription of a cell exposed to a stress using the lentiviral vector.

A method of differentiating nonhuman primate induced pluripotent stem cells (iPSCs) to oligodendrocytes is provided. The method may include providing a modified vector comprising a piggyBac vector backbone that expresses one or more differentiation factor genes, including SOX10, OLIG2, and NKX6-2. The piggyBac vector backbone may comprise one or more terminal inverted repeats and one or more transposase recognition sites configured to interact with a piggyBac transposase to mediate genomic integration at TTAA or noncanonical target sequences. The vector may further include a selectable marker and a promoter for regulated expression. The modified vector may be introduced into nonhuman primate iPSCs, for example from Macaca fascicularis, by electroporation and used in a feeder-free differentiation workflow. The resulting differentiated cells may be identified by expression of oligodendrocyte-associated markers including O4, O1, PDGFRA, MOG, GALC, CNPase, and myelin basic protein (MBP).

The present invention provides a genome modification method and a genome modification kit that can efficiently modify two or more alleles and modify relatively large regions. [Solution] A genome modification method for modifying two or more alleles of a chromosome genome, comprising: (a) introducing the following (i) and (ii) into a cell containing the chromosome; (i) a genome modification system comprising a sequence-specific nucleic acid cleavage molecule that targets a target region of the chromosome genome, or a polynucleotide encoding the sequence-specific nucleic acid cleavage molecule; (ii) two or more selection marker donor DNAs having mutually different selection marker genes (the number of types of selection marker donor DNAs is equal to or greater than the number of alleles to be modified); and (b) selecting the cell based on all the selection marker genes possessed by the two or more selection marker donor DNAs.

Disclosed arc vectors including i) a first region of nucleic acid sequence comprising a gene of interest, a telomerase occupancy site, and 5' and 3' ends; and ii) a spacer region that is less than 1000 bp links the 5' and 3' ends of the first region of nucleic acid sequence. Vectors may also include a bacterial replication origin or a nucleic acid sequence encoding a RNA selectable marker. Vectors may be double-tranded, linear, close-ended nucleic acid vector. Also disclosed are methods for producing double-stranded, linear, close-ended nucleic acid vectors.

Inventions disclosed herein relates to vectors and expression systems for producing heteromeric recombinant proteins such as monoclonal antibodies. Vectors and expression systems disclosed herein are based on the finding that the selectable marker glutamine synthetase can be divided into two fragments at selected amino acid positions of the glutamine synthetase polypeptide, and the two fragments can internet and / or associate to form a monomer and then a functional multimeric glutamine synthetase protein.

The present invention provides: a novel marker for genetic recombination of Starmera bombicola; and a method for using the same. The present invention pertains to: a Starmerella bombicola mutant strain in which expression of an orotate phosphoribosyltransferase-like protein or an equivalent protein is suppressed or the protein is inactivated; a uracil auxotrophic selectable marker comprising the gene; and a method for producing a transformant of Starmerella bombicola using the selectable marker.

The invention discloses a brassica napus clubroot resistance molecular selective marker primer and application thereof. Nucleotide sequences of the primer are shown as SEQ ID NO.2 and SEQ ID NO.3. The invention further discloses a method for detecting the clubroot resistance molecular selective marker primer for the brassica napus. The invention also provides an application of the primer in breeding of brassica napus. In the technical scheme adopted by the invention, whether the brassica napus contains the Crr1 gene derived from the Chinese cabbage or not can be clearly judged through different sizes and positions of strips by using agarose with the concentration of 2% for 15 minutes, the method is simple and rapid, the detection cost is low by using each site of each sample, a special instrument is not needed, and if the method has the characteristics of simplicity and convenience in operation and cost saving, the method can be widely applied to the detection of the Crr1 gene derived from the Chinese cabbage. Popularization and application are facilitated.

Disclosed are a multi-vector expression system encoding precursor mRNA molecules and precursor mRNA trans-splicing molecules (PTM), and a cultured eukaryotic recombinant host cell comprising the system. Furthermore, disclosed are a method for recombinantly expressing one or more target proteins in a cultured eukaryotic host cell line, and a method for screening a eukaryotic host cell recombinantly expressing a target protein in vitro, which employ the multi-vector expression system of the present application.

The present invention relates to a mammalian expression vector comprising a polynucleotide encoding bacterial glutamine synthetase as a selection marker, as well as a nucleic acid encoding said bacterial glutamine synthetase and a mammalian host cell. In particular, said bacterial glutamine synthetase is from a bacterium of the order Enterobacteriale and the family Morganellaceae, preferably said bacterial glutamine synthetase is a glutamine synthetase from the genus Providencia, optionally further comprising a mutation at position E130 and / or F226 and / or R345A. The present invention further relates to a method for preparing a mammalian cell stably expressing a protein and / or a non-coding RNA of interest, and a method for producing a protein in a mammalian cell using said bacterial glutamine synthetase.

Methods and compositions are provided for modifying the genome of Bacillus sp. cells without the use of a selectable marker and without the use of a guided Cas endonuclease. The disclosure includes methods for integrating donor DNA sequences into the genome of a Bacillus sp. cell without the use of a selectable marker and without the use of Cas endonucleases into said genome, as well as methods for deleting genes of interest and / or providing point mutations into the genome of Bacillus sp. cells.

The application discloses a riboswitch element responding to vitamin B12 and construction and application of a vitamin B12 biosensor based on the riboswitch element, and belongs to the fields of genetic engineering technology, molecular detection and biosensing. The biosensor comprises a riboswitch element responding to vitamin B12 and a selectable marker gene. An effective relationship between vitamin B12 concentration in a bacterium and expression of the marker gene is established by using the sensor. The biosensor can be used for selecting a high-yield vitamin B12 strain in a relatively fast and easy way, and has an important application prospect in detection and screening of vitamin B12-producing microorganisms.

Methods for scarless genome editing are provided, particularly scarless genome modification by using a homology-directed repair (HDR) step to genetically modify a cell and remove an undesired sequence.SOLUTION: Provided is a method for scarless editing of genomic DNA of a cell comprising the following steps. The method comprises the steps of: (a) performing a first Cas9-mediated homologous recombination repair (HDR) step; (b) isolating genetically modified cells based on a positive selection of at least one selectable marker; (c) performing a second Cas9-mediated homologous recombination repair (HDR) step; and (d) isolating genetically modified cells containing the intended editing based on a negative selection of at least one selectable marker, wherein an expression cassette encoding the at least one selectable marker has been deleted.SELECTED DRAWING: Figure 1A

Provided herein are polynucleotides, vectors, complexes, compositions, systems, kits, methods and uses for enriching for gene editing in a cell. Some aspects of the disclosure relate to the use of a polynucleotide cassette comprising a coding sequence of a selection marker gene that is disrupted by a synthetic intron sequence to prevent gene expression of the selection marker. The synthetic intron further comprises a defective splice site that is correctable by a base edit such that when the splice site is corrected by a gene editor, the intron is removed by the endogenous slicing system of the cell and expression of the selection marker gene can occur. Methods described herein screen for the presence of active gene editors within the cell by subjecting the cell to the selection pressure of the selection marker.

The invention relates to a multi-gene pyramiding breeding method suitable for animal populations, the method adopts a minimum rotation maximum complementation (mRMC) gene pyramiding method, compared with the prior art, all individuals in the population can stably carry n target genes (ideal populations) at the same time by needing fewer generations, the number of generations is reduced, the number of generations is reduced, the number of generations is reduced, and the number of generations is reduced. And it is ensured that the group still contains n families. According to the method, the gene polymerization efficiency is effectively improved, and the method shows superiority in the aspects of controlling the population inbreeding level, maintaining the population genetic diversity, maintaining the background character genetic progress and the like. The method. The method can be combined with breeding technologies such as genome selection, marker-assisted selection, comprehensive selection indexes and the like, provides an efficient, controllable and programmable general technical framework for polygene pyramiding breeding of livestock and poultry such as pigs, cattle, sheep, chickens and the like, and has important application value for accelerating the molecular design breeding process and breeding new varieties with high resistance and high yield.

The invention discloses a gene modification method of a fusion cytokine expression element and an immunotherapy application of the fusion cytokine expression element, and relates to the technical field of gene engineering and tumor immunotherapy. Comprising the following steps that A, a preset site is selected in a genome of a host cell, a first construct AL-B-C-B-AR is provided, AL is a first homologous arm located on the upstream of the preset site, AR is a second homologous arm located on the downstream of the preset site, B is an I-SceI incision enzyme recognition site, and C is a selection marker capable of being screened in the forward direction; inserting the B-C-B sequence into the predetermined loci by homologous directional integration to form a replaceable landing frame; wherein the preset site is preferably a safe port site and a non-essential intron segment of a target functional gene, the length of the homologous arm is preferably one hundred to one thousand bases, and after integration, the correctness of the 5'end and the 3 'end is confirmed through PCR breakpoint amplification and sequencing at the same time. The whole body exposure and toxicity are reduced.

The invention discloses a random multi-copy integration tool based on an rNTS site. The invention also discloses a method for carrying out multi-copy gene integration and screening in pichia pastoris by using the random multi-copy integration tool. The invention also discloses an application of the random multi-copy integration tool in construction of a pichia pastoris engineering strain for producing zeaxanthin. Appropriate optimization can be carried out according to specific requirements to achieve the purpose of screening different strains, rNTS no-load plasmids represented by different fluorescent proteins are adopted, random multi-copy integration of multiple heterologous genes can be carried out at the same time, and powerful support is provided for construction of a complex gene network. According to the invention, the copy number range is determined by using the fluorescence ratio generated by the fluorescent proteins of which the wavelengths do not influence each other, the accuracy and reliability are high, and the detection is simple and rapid; the simultaneous mutation of LoxP sites lox66 and lox71 avoids the potential interference of the residue of the selective marker gene on the subsequent experiment.

The present invention is concerned with a human CD20 protein with modifications to one or more intracellular domains sufficient to reduce or abrogate intracellular signalling when attached to or associated with the membrane of the cell such as (e.g.) a T-cell, a natural killer cell, a B-cell, a myeloid cell, a pluripotent stem cell and a haematopoietic stem cell. The present invention further contemplates a nucleic acid encoding a modified human CD20 protein described herein, and to the utility of the modified human CD20 protein as a safety switch in chimeric antigen receptor T-cell therapy or as a selection marker for gene therapies.